Nodule CO2 fixation via PEPC provides malate for bacteroids and oxaloacetate for N assimilation. The process is therefore of central importance for efficient nitrogen fixation. Nodule CO2 fixation is known to depend on external CO2 concentration. The hypothesis of the present paper was that nitrogen fixation in alfalfa plants is enhanced when the nodules are exposed to elevated CO2 concentrations. Therefore nodulated plants of alfalfa were grown in a hydroponic system that allowed separate aeration of the root/nodule compartment that avoided any gas leakage to the shoots. The root/nodule compartments were aerated either with a 2500 microl l(-1) (+CO2) or zero microl l(-1) (-CO2) CO2-containing N2/O2 gas flow (80/20, v/v). Nodule CO2 fixation, nitrogen fixation, and growth were strongly increased in the +CO2 treatment in a 3-week experimental period. More intensive CO2 and nitrogen fixation coincided with higher per plant amounts of amino acids and organic acids in the nodules. Moreover, the concentration of asparagine was increased in both the nodules and the xylem sap. Plants in the +CO2 treatment tended to develop nodules with higher %N concentration and individual activity. In a parallel experiment on plants with inefficient nodules (fix-) the +CO2 treatment remained without effect. Our data support the thesis that nodule CO2 fixation is pivotal for efficient nitrogen fixation. It is concluded that strategies which enhance nodule CO2 fixation will improve nitrogen fixation and nodule formation. Moreover, sufficient CO2 application to roots and nodules is necessary for growth and efficient nitrogen fixation in hydroponic and aeroponic growth systems.